1. Field of the Invention
The invention relates to methods and compositions useful for enhancing iron release from ferritin, including methods and compositions useful for treatment of iron overload.
2. Description of the Related Art
The Biochemistry of Iron Storage
Iron biochemistry is complex and highly regulated. Several carriers are involved in iron transport, including the protein transferrin. The majority of stored iron in bodily tissues is in the protein ferritin, which has the same function in animals, plants and bacteria. In healthy individuals, approximately 1–12% of total body iron is stored in ferritin. In patients with iron overload, this value is much greater.
Ferritin is a 24 subunit protein that forms a large cavity (8 nm) that concentrates up to 4,500 iron atoms as a solid mineral, although the “nano-rock” is normally found with closer to 30% of this value. In animals, ferritin is present both in serum and in tissues, especially in the liver and in bone marrow where it serves as an iron reserve for the production of hemoglobin; the serum form of ferritin, which contributes little to overall iron storage, nevertheless finds clinical use as a reporter of body iron levels. Ferritins occur in animals as approximately 25 distinct isoforms depending on their proportions of the two primary subtypes of ferritins, H or L. These distinct subtypes also differ in their rates and mechanisms of iron oxidation, core formation and iron turnover. The active sites in H-type ferritin rapidly increase, by a factor of 104, the rate at which the mineral core is created, at the expense of producing some hydrogen peroxide. L-type ferritin subunits lack the residues necessary to form an active site. See, e.g., Theil, E. C. in Handbook of Metalloproteins, (A. Messerschmidt et al., eds.), John Wiley & Sons, Chichetser, pp. 771–781 (2001); Andrews, S. C. Adv. Microb. Physiol. 40:281–351 (1998); Chasteen, N., Harrison, P., J. Struct. Biol. 126:182–194 (1999); Harrison, P., Arosio, P., Biochem. et Biophys. Acta 1275:161–203 (1996).
Iron uptake by ferritin results in the formation of an oxidized Fe(III) mineral. Iron release from ferritin can be effected through treatment with various reductants in vitro and in vivo. The size of the reductant has no effect on the rate of reductive release of iron (Watt G. D., et al., Proc. Natl. Acad. Sci USA. 82:3640–2643 (1985); Watt G. D., et al., Proc. Natl. Acad. Sci. USA 85:7457–7461 (1988)).
Structural studies on ferritin and ferritin mutants have helped to clarify the mechanism of iron release. Structure at the junction of three subunits in ferritin creates “pores” through which iron exits from the hydrated ferric oxide core. There are eight three-fold junctions in each ferritin molecule. Structural studies of a mutant form of ferritin (H-L134P) showed a region that became disordered in the crystal structure as a result of a mutation that locally disrupted the structure of the protein at the three-fold junctions (Takagi H., et al. (1998) J. Biol. Chem. 273:18685). Measurements of iron release rates showed that the iron can be cleared from the mutant proteins, in test environments, within five minutes compared to 150 minutes in the closed ferritin protein.
Another structural study focused on a larger set of mutants that were expected to affect the geometry or charge of conserved amino acids at the three-fold junctions (Jin, W. et al., Biochemistry, 40: 7535–7532 (2001)). Alterations of a conserved hydrophobic pair, a conserved iron pair, and a loop at the ferritin pores to which no other function had been assigned, all increased iron exit by 3–30 fold, with no apparent effect on ferritin assembly except for a slight decrease in volume. The pores in these mutants appear to be “locked” in the “open” position.
Iron Overload
Iron overload is a complication of the treatment, by chronic transfusion, of a number of genetic diseases associated with inadequate red cell production (anemias) and of other genetic diseases that lead to excessive iron absorption from the diet. Two relatively common anemic conditions that can result in iron overload from hypertransfusion are sickle cell disease and thalassemia. Iron overload is a serious condition that can cause fatal cardiac damage or stroke if left untreated. See, e.g., Golden, C. et al., Curr. Opin. Hematol., 5(2):89–92 (1998). Since the genes responsible for the diseases that lead to iron overload (either congenitally or indirectly from transfusion treatment) are very common in the population, the combined frequency of carriers for the diseases that can involve iron overload approaches 20% in North American and European populations. In Africa, Southeast Asia, and the Mediterranean, where malaria is endemic, and among the descendents of such populations world-wide, single gene mutation frequencies are as high as 10%. See Ashley-Koch, A. et al., Amer. J. Epid., 51(9): 839–45. Worldwide, hundreds of thousands of people suffer from iron overload and a billion suffer from other abnormalities of iron homeostasis.
Current Treatments
Currently, iron overload from excess absorption is treated by regular phlebotomy (bleeding), while iron overload from hypertransfusion is treated by long periods of intravenous or subcutaneous administration (up to 8 hours/day for at least 5 days/week) of an iron chelator. Clinically used chelators are designed to bind extracellular iron and to pull the iron from intracellular sites. Such an indirect approach leads to the necessity of the long exposure periods.
In spite of the many efforts to develop new and enhanced chelation treatments for iron overload, the best treatment available remains intravenous or subcutaneous administration of desferrioxamine (e.g., Desferal®). Although desferrioxamine treatment is effective and safe, a number of side effects have been observed, including inhibition of growth, bone abnormalities, retinal damage, ocular toxicity and ototoxicity (Hoffbrand, A. V., Curr. Op. Hematol. 2:153–158 (1995)). In addition, desferrioxamine administration may also lead to an allergic response through the activation of mast cells (Magro, A. M., Brai M., Immunology 49:1 (1983); Shalit, M., et al., J. Allergy Clin. Immunol. 88:854 (1991); Lombardo, T., et al., Am. J. Hematol. 51:90 (1996)). This allergic response can lead to pain and irritation at the point of injection.
More limiting to the current therapeutic approaches is the issue of patient compliance. The necessity of slow infusion of the chelator over a period of 8–24 hours a day makes patient compliance a serious issue in the treatment. The occurrence of side effects, especially allergic reaction, irritation or pain, encourages lack of compliance. Another problem with the current method of treatment is that such long-term treatment is also expensive and impractical for most of the world's population. The drug itself, desferrioxamine, is also expensive.
Richardson and Ponka have proposed requirements for the development of improved iron chelators for the treatment of iron overload (Richardson D. R., Ponka P., Am. J. Hematol. 58:299–305 (1998)). Briefly, the chelator should be: (1) biospecific, having high affinity for iron over other physiologically important cations, and for stored iron rather than iron functioning in important enzymes such as hemoglobin; (2) bioavailable, and preferably orally available; (3) stable to degradation by enzymes; (4) biocompatible, with minimal side effects; (5) highly effective at promoting iron excretion; and (6) readily and inexpensively synthesized.
Following these criteria, major efforts have been undertaken to develop new iron chelators that are orally available for the treatment of iron overload. Desferrioxamine, for instance, can be administered orally but oral administration greatly reduces its efficacy as compared to either intravenous or subcutaneous administration (Katramis C., et al., Lancet 1:51 (1981)). Newer chelating agents are also under consideration, but of the siderophore class of chelators, desferrioxamine has proved to be the most effective for treating iron overload (Richardson D. R., Ponka P., Am. J. Hematol. 58:299–305 (1998)). Also under consideration is the α-keto-hydroxypyridone chelator 1,2-dimethyl-3-hydroxypyrid-4-one, variously known as deferiprone, L1 or CP20. However, recent clinical data have shown that deferiprone is not completely effective at managing iron overload and may worsen hepatic fibrosis (Olivieri, N. F. & Brittenham, G. M., Blood 89:739 (1997); Olivieri, N. F., et al., N. Engl. J. Med. 339:417–423 (1998)).
While the references mentioned above for suggested improvements to chelators are relevant to understanding the problem of iron overload, they do not teach a solution to the problem of treatment of iron overload. Among other disadvantages, none of the references teaches how to enhance the rate of iron release from native ferritin, such that chelation of released iron is facilitated. Thus, there is a clear need for more efficacious methods of treating iron overload, in addition to methods and reagents useful for the identification and characterization of compounds which enhance iron release from ferritin. The present invention addresses these and other shortcomings of the prior art.